Glomax microplate luminometer

Cells were seeded at 10⁵ cells per well in 24-well plates in DMEM containing 10% FBS at one day prior to transfection. HCT116-TetOnIII cells were transfected in triplicate with PEI-MAX (Polysciences, USA), 200 ng of dual luciferase target reporter plasmid (Supplementary Figure S2) and 1–30 ng of TuD RNA expression plasmid. HCT116-TetOn-TuD141/200c cells and HCT116-TetOn-TuDNC cells were transfected in triplicate with PEI-MAX and 200 ng of dual luciferase target reporter plasmid. We performed all dual luciferase reporter assays at 48 hours after transfection using the Glomax microplate luminometer (Promega).

Human A549 and avian DF-1 cells were transfected with plasmids for the expression of Giza virus PB2, PB1, PA, and NP proteins. To test the PB2-A84T and -D146G mutations, the wild-type PB2 protein expression plasmid was substituted with plasmids expressing Dakahlia PB2-A84T or Giza PB2-D146G, respectively (note that the Dakahlia and Giza PB2 genes differ by two synonymous nucleotide replacements, but encode identical PB2 proteins). Cells were also transfected with pPol-I-NP(0)Luc2(0) (for A549 cells) or pPol-IGG-NP(0)Fluc(0) (for DF-1 cells), which express the firefly luciferase reporter protein from a virus-like RNA transcribed by the human or avian polymerase I promoter, respectively. Plasmid pRL-TK (Promega, Madison, WI) served as an internal control for the dual-luciferase assay. Transfected cells were incubated for 24 h at 33 °C and 37 °C (human A549 cells), or at 39 °C (avian DF-1 cells). Luciferase activity was measured by using the Dual-Glo luciferase assay system (Promega) on a Glomax microplate luminometer (Promega) according to the manufacturer’s instructions. The results of two experiments (both carried out in duplicate) were compared using one-way ANOVA, followed by Tukey’s Post-hoc test. We considered the results significant at p < 0.05.

The HepG2 cells were seeded in 12-well-plates at a concentration of 2 × 10⁵ cells/well for 24 h, and then, transfected with a promoter-reporter plasmid plus vectors containing the gene of interest by Lipofectamine™ 3000 (Invitrogen). The Renilla luciferase reporter plasmid was used as the internal control of transfection efficiency. After transfection for 48 h, the luciferase activity was measured using a GloMax microplate luminometer (Promega).
All vectors used in this study were purchased from GenePharma (Shanghai, China). The restriction enzymes, different modification enzymes, and T4-DNA ligase were purchased from MBI Fermentas (Ontario, Canada).

Cell viability was assessed by luminescent cell viability assay with CellTiter-Glo (Promega, Madison, WI, USA) according to the manufacturer’s protocol. This assay determines the number of viable cells in culture based on quantitation of ATP, an indicator of metabolically active cells. Briefly, MODE-K cells were plated into 96-well plates and treated as described under cell culture. At the end of the incubation period for 12 h with CO-RMs on day 3, an equal volume of the luminescent substrate and lysis buffer mix from the assay kit was added. The mixture was transferred to an opaque 96-well plate, and luminescence was recorded using a GloMax Microplate Luminometer (Promega). The index of cellular viability was calculated as the percentage of luminescence with respect to untreated control cells.